Toll-Like Receptors and Skin Cancer.

tami&g odd

IMMUNOLOGY

OPINION ARTICLE

published: 31 March 2014
doi: 10.3389/fimmu. 2014. 00135

Toll-like receptors and skin cancer

Erin M. Burns and Nabiha Yusuf *

Department of Dermatology, University of Alabama at Birmingham, Birmingham, AL, USA
'Correspondence: nabiha@uab.edu
Edited and reviewed by:

Christophe M. Filippi, Genomics Institute of the Novartis Research Foundation, USA

Keywords: Toll-like receptor, non-melanoma skin cancer, BCC, SCC, melanoma, innate immunity

The skin, the largest organ in the body,
provides the first line of defense against
the environment both as a physical barrier
and as a key immunological component.
Toll-like receptors (TLRs) serve as signal-
ing molecules that recognize pathogen-
associated molecular patterns (PAMPs)
as well as damage-associated molecular
patterns (DAMPs), and are expressed by
various skin cells including keratinocytes
and melanocytes, which are the main cell
types involved in both non-melanoma
and melanoma skin cancers. TLRs induce
inflammatory responses meant for clear-
ing pathogens, but can ultimately con-
tribute to skin carcinogenesis. In contrast,
TLR agonists, specifically targeting TLR7,
8, and 9, have been successfully used as
therapeutics for melanoma and basal cell
carcinoma (BCC), functioning by recruit-
ing dendritic cells and inducing T-cell
responses. Here, we discuss the role TLRs
play in skin carcinogenesis as well as the use
of TLRs as targets for skin cancer treatment
options.

SKIN AND TLRs

Non-melanoma skin cancer (NMSC)
includes BCC and squamous cell carci-
noma (SCC). With over 3.5 million new
diagnoses annually, NMSC is the most
common cancer in the United States (1).
Risk factors for developing NMSC include
ultraviolet (UV) light exposure, skin color,
sunburns, age, and immunosuppressive
status (2). NMSCs account for over 3,000
deaths each year (3) and also contribute to
over S 1 .4 billion annually for the treatment
and management of these skin tumors (4).
Melanoma contributes to approximately
5% of all skin cancer diagnoses, with 76,000
new cases diagnosed in 2012 (5). Impor-
tantly, melanoma leads to over 9,000 deaths
annually, which accounts for the major-
ity of skin cancer deaths. Risk factors for
melanoma include UV exposure, sunburn,

nevi, immunosuppressive status, and fam-
ily history.

The most common treatments for
SCC include excision, Mohs micrographic
surgery, and cryosurgery, which, when
the lesion is detected early and promptly
removed, are effective and cause minimal
damage (2). If left untreated, the tumors
are able to grow exponentially or metasta-
size, leading to more invasive procedures.
For melanoma, surgical excision is the
most common treatment, with recent pref-
erences for Mohs surgery (5). However,
in the case of recurring lesions or lesion
patches, surgery may not be an option
due to extensively damaged skin or lack of
tissue for removing clear margins, result-
ing in the need for alternative treatment
options.

The skin is the largest organ in the body
and contains three major cell types, which
include melanocytes, Langerhans cells, and
keratinocytes. Keratinocytes are the major
cell type of the epidermis and provide
defense against the environment both as a
physical barrier and a key component of
the innate immune response (6, 7). Epi-
dermal keratinocytes, as the outmost envi-
ronmental barrier, are responsible for the
production of antimicrobial peptides (8),
which are up-regulated by various stim-
uli through both the mitogen-activated
protein (MAP) kinase and nuclear fac-
tor (NF) kappaB pathways (9). TLRs are
expressed by various skin cells includ-
ing keratinocytes and melanocytes (10),
which are the main cell types involved in
both non-melanoma and melanoma skin
cancers. Human keratinocytes have been
shown to express TLRs 1-6 and 9 (10-14).
Recently, it has been reported that TLR2-5,
7, 9, and 10 are constitutively expressed in
human melanocytes (15).

Toll-like receptors serve as signal-
ing molecules that recognize PAMPs, or
pathogen-associated molecular patterns, as

well as DAMPs and thus, activate the innate
immune response through the transcrip-
tion factor NF-kB ( 16). The 10 human TLR
family members are characterized by the
leucine-rich repeat domain content in both
their extracellular region and the intracel-
lular Toll-IL-1 receptor (TIR) domain (17),
which can therefore interact with adaptor
molecules that contain appropriate adaptor
molecules (18).

Toll-like receptors have been demon-
strated to be important for both innate
immune response specificity (19, 20) as
well as for adaptive immune responses such
as dendritic cell maturation and costim-
ulatory molecule expression and the pro-
motion of Th-1 cell-mediated responses
through increased production of IL-12 by
activated TLRs on dendritic cells (2 1 , 22) . It
also has been reported that innate inflam-
matory responses localized to the epider-
mis may be affected by TLR expression in
human melanocytes (23). TLRs are acti-
vated in melanocytes, as a consequence of
the inflammatory response to tissue injury,
sunburn or skin infection, and constitute a
natural defense to recruit innate immune
cells.

TLR STIMULATION AND SKIN
CARCINOGENESIS

Besides their function of recognizing
exogenous PAMPs, TLRs also recognize
endogenous ligands, which are often
referred to as alarmins and function to
recognize cell or tissue damage and alert
the innate and adaptive immune systems
(24, 25). Expression association studies
have revealed potential functions of TLR
endogenous ligands in tumorigenesis. For
example, high-mobility group box-1 pro-
tein (HMGB1) can function as a DAMP
and is released in response to tissue or cel-
lular damage. It is over-expressed in several
human neoplasms including lung, pancre-
atic, breast, liver, and colorectal cancers,

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TLRs and skin cancer

and, importantly, melanoma (26). HMGB1
is either passively released by injured or
necrotic cells (27) or actively secreted by
monocyte/macrophages, neutrophils, and
dendritic cells [reviewed in Ref. (28)].

With the exception of TLR3 that sig-
nals through Toll/IL- 1R domain containing
adaptor inducing IFN (TRIF), TLRs signal
through myeloid differentiation factor 88
(MyD88). TLR signaling has been reviewed
extensively elsewhere (29). MyD88 is an
adaptor protein that is ultimately respon-
sible for initiating NF-kB activation (30),
and therefore the amplification of inflam-
mation and the promotion of tumor devel-
opment (31). Importantly, chronic inflam-
mation has been linked to tumor devel-
opment in animal models of both sponta-
neous and chemically induced carcinogen-
esis (32, 33).

Tumor cells expressing TLRs may
be able to evade immune surveillance
processes, thus promoting tumor develop-
ment. The activation of TLR4 and sub-
sequent signaling molecules have been
shown to upregulate immunosuppressive
cytokines such as IL-10 as well as pro-
inflammatory cytokines and chemokines
including IL-6, IL-18, and TNF-a, which
have been shown to contribute to tumor
development, growth, and even metasta-
sis (34). In human melanoma A375 cells,
the inhibition of TLR4/MyD88 signal-
ing effectively decreased both VEGF and
IL-8 levels with paclitaxel and icariside
II combination treatment (35). TLR2-4
are expressed and up-regulated in sev-
eral human metastatic melanoma cell lines
(36), with recent data indicating that
melanoma cells also express TLR7, 8,
and 9 (37), which are abnormally up-
regulated in cells from melanoma biopsies
(38). The over-expression of TLR4 within
melanoma tumors triggers an inflamma-
tory response leading to tumor devel-
opment (39). TLR9 activation has also
been shown to enhance invasion as well
as promote proliferation in several can-
cer cell lines via NF-kB and Cox-2 acti-
vation (40), as well as the secretion of
IL-8 and IL-la (41), and TGF-fi (42).
Recent studies in head and neck cancer
have revealed that TLR3 expression and sig-
naling affects the migration and metasta-
tic potential of tumors as evidenced in
oral SCC by inducing CCL5 and IL-6
secretion (43).

Importantly, TLR inhibition can exert
anti-cancer effects. TLR4 pathway inhi-
bition reversed tumor-mediated suppres-
sion of both natural killer cell activity
as well as T-cell proliferation in vitro
and in vivo, resulting in increased tumor
latency and survival of tumor-bearing mice
(44). TLR2 plays an important role in
the induction of tumor regression, which
has been demonstrated in a mouse model
of glioblastoma multiforme where block-
ing HMGB1 -mediated TLR2 signaling via
tumor-infiltrating myeloid DCs resulted in
a loss of therapeutic efficacy (45).

TLR3 activation on immune cells results
in anti-cancer activities, where T cell-
mediated responses are promoted (46).
Specifically, upon stimulation with TLR3
agonist poly(LC), CD8 T cell responses
are enhanced, leading to the production
of IFNy and TNF-a and ultimately, the
generation of memory CD8 T cells.

TLR-TARGETED THERAPY

Although TLR expression on tumor cells
may allow tumors to evade surveillance,
TLRs are also considered to be targets for
anti-cancer interventions that result in the
recognition and ultimate destruction of
tumor cells using a tolerant immune sys-
tem. This idea is further illustrated by the
fact that recent studies have demonstrated
a dual nature of immune responses in the
context of cancer therapies, highlighting
the importance of considering conditions,
TLR targets, and combinations of immune
interventions and TLR ligands (47).

There are studies and case reports that
show that 5% imiquimod cream treatment
is an effective therapeutic option for actinic
keratosis (AK), BCC, Bowen's disease, and
lentigo maligna (48-53). The mechanism
of action of imiquimod is through the
activation of TLR7 (54), and imiquimod
has been approved to treat both prema-
lignant actinic keratoses, and malignant
superficial BCC (55). The mechanism may
also involve Thl -response promotion, the
recruitment of macrophages, anti-tumor
cytotoxic CD8 T cells, and NK cells to the
lesion, as well as induce apoptosis of tumor
cells (55, 56). Imiquimod has also been
shown to induce IFN-a and IL-12 pro-
duction, resulting in a heightened immune
response (49, 57, 58). The suggested mech-
anism for exertion of anti-tumor effects
on UVB-induced SCC by imiquimod is

through the activation of Thl7/Thl cells
as well as cytotoxic T lymphocytes (59).
Five percent topical imiquimod has been
effective in several clinical trials (49, 53, 57,
60). The related drug, resiquimod, has been
demonstrated as a safe and effective topical
intervention for AK and is a potential treat-
ment option for patients who have large
patches of AK(61).

Several cancer types including
melanoma have been successfully treated
with Taxol, CpG, or otherTLR ligands (62,
63). PF35 12676, a synthetic CpG ODN,
uses a TLR9-targeted approach to effec-
tively treat BCC (64). TLR 7 and 8 agonists
activate a pro-inflammatory response for
SCC treatment (65). Additionally, IL-1,
6, 8, and 12 modulation along with a
promotion of a Thl -response have been
shown to exert anti-tumor and antiviral
behavior (65).

Previous studies have demonstrated
TLR3 agonists to be promising adjuvants
for cancer vaccines, especially in regards
to their immunostimulatory properties
(46). A recent study has demonstrated
that human melanoma cells express TLR3,
which in combination with TLR3 agonists,
results in tumor cell death via caspase
activation when cells are pretreated with
cycloheximide or IFN-a (38), suggesting
that TLR3 agonists may be multifunctional
adjuvants offering more clinical treatment
options. Therefore, TLRs and their signal-
ing pathways may be potential therapeu-
tic targets to control tumor progression,
especially in diseases such as cutaneous
malignant melanoma, which is an aggres-
sive tumor that is not effectively managed
with current treatments (66).

It is important to note that, especially
in the case of TLR7 agonists such as
imiquimod and resiquimod, though quite
effective when applied topically to AKs
and BCCs, systemic therapeutic interven-
tions have not been as successful. This
TLR tolerance has previously been demon-
strated with TLR4 agonists, which resulted
in decreased NF-kB activation (67). The
suggested mechanism for TLR7 tolerance is
the diminished capacity for IL-12 secretion
as well as IFN-a secretion by plasmacytoid
DCs (68). Recent studies have found that
local and systemic TLR-targeted therapies
have different modes of action and require
further investigation, especially into the
timing and dosage of treatments to reach

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TLRs and skin cancer

maximum efficacy without inducing TLR
tolerance (69).

CONCLUSION

In summary, TLRs are an important
immunological component expressed by
keratinocytes and melanocytes, which are
the main cell types involved in both
non-melanoma and melanoma skin can-
cers. TLRs induce inflammatory responses
meant for clearing pathogens, but their
activation can also potentiate chronic
inflammation, which can ultimately con-
tribute to skin carcinogenesis. In contrast,
TLR agonists, specifically targeting TLR7,
8, and 9, have been successfully used as
therapeutics for melanoma and BCC, func-
tioning by recruiting dendritic cells and
inducing T-cell responses. It is important
to consider local versus systemic applica-
tions of TLR therapies and the balance
between efficacy and inducing TLR tol-
erance. TLR3 agonists have been shown
to be well-tolerated and effective in both
directly killing cancer cells and directing
immune responses in melanoma. TLR-
targeted therapies may be potential treat-
ment options for large or reoccurring skin
tumors that may be difficult to treat with
surgery or for other skin tumors that are
not responsive to current therapies.

ACKNOWLEDGMENTS

This work was supported by NIH Can-
cer Prevention and Control Training Grant
(R25CA47888) to Erin M. Burns.

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Received: 10 January 2014; accepted: 17 March 2014;

published online: 31 March 2014.

Citation: Burns EM and Yusuf N (2014) Toll-like

receptors and skin cancer. Front. Immunol. 5:135. doi:

W.3389/fimmu.2014.00135

This article was submitted to Immunological Tolerance,
a section of the journal Frontiers in Immunology.
Copyright © 2014 Burns and Yusuf. This is an open-
access article distributed under the terms of the Creative
Commons Attribution License (CC BY). The use, dis-
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provided the original author(s) or licensor are credited
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Frontiers in Immunology | Immunological Tolerance

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